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    GraphPad Prism Inc graphpad prism 6 0 software
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    Graph Pad Software Inc graphpad prism 6 0 software
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    GraphPad Prism Inc statistical analysis graphpad prism 6
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    GraphPad Prism Inc statistical analysis graphpad prism 6 0
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    GraphPad Prism Inc graphpad prism 6 02 software
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    GraphPad Prism Inc graphpad prism 6 0
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
    Graphpad Prism 6 0, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 91/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GraphPad Prism Inc graphpad prism 6 05 software
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    GraphPad Prism Inc program graphpad prism 6
    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the <t>GraphPad</t> Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
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    Modulation of programmed cell death ligand-1 (PD-L1) levels by chemotherapeutic drugs in non-small-cell lung cancer (NSCLC) cell lines. ( A ) A549, Calu-6, H292, and H322 cells were cultured in complete medium and after 24 h, the cells were collected and PD-L1 expression was evaluated by flow cytometry and quantified as molecules of equivalent fluorochrome (MEF). ( B ) Cancer cells were treated with increasing concentrations of the indicated drugs for 72 h. IC 50 values ± SD were calculated using <t>GraphPad</t> Prism 6.00 software. ( C ) NSCLC cells were treated with gemcitabine, cisplatin, paclitaxel, vinorelbine, or pemetrexed at IC 50 concentrations. After 3 days, PD-L1 levels were analyzed by flow cytometry. ( D ) Confocal immunofluorescence analysis of PD-L1 expression in H322 cells maintained in the absence or in the presence of 100 nM pemetrexed for 72 h; green fluorescence indicates the positivity to PD-L1. The nuclei were stained with Draq5 (red fluorescence). Scale Bar: 10 μm. Data in ( A ) and ( B ) are mean values ± SD of three independent experiments. Results in ( C ) and ( D ) are representative of two independent experiments.
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    GraphPad Prism Inc statistics graphpad prism 6
    Modulation of programmed cell death ligand-1 (PD-L1) levels by chemotherapeutic drugs in non-small-cell lung cancer (NSCLC) cell lines. ( A ) A549, Calu-6, H292, and H322 cells were cultured in complete medium and after 24 h, the cells were collected and PD-L1 expression was evaluated by flow cytometry and quantified as molecules of equivalent fluorochrome (MEF). ( B ) Cancer cells were treated with increasing concentrations of the indicated drugs for 72 h. IC 50 values ± SD were calculated using <t>GraphPad</t> Prism 6.00 software. ( C ) NSCLC cells were treated with gemcitabine, cisplatin, paclitaxel, vinorelbine, or pemetrexed at IC 50 concentrations. After 3 days, PD-L1 levels were analyzed by flow cytometry. ( D ) Confocal immunofluorescence analysis of PD-L1 expression in H322 cells maintained in the absence or in the presence of 100 nM pemetrexed for 72 h; green fluorescence indicates the positivity to PD-L1. The nuclei were stained with Draq5 (red fluorescence). Scale Bar: 10 μm. Data in ( A ) and ( B ) are mean values ± SD of three independent experiments. Results in ( C ) and ( D ) are representative of two independent experiments.
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    Modulation of programmed cell death ligand-1 (PD-L1) levels by chemotherapeutic drugs in non-small-cell lung cancer (NSCLC) cell lines. ( A ) A549, Calu-6, H292, and H322 cells were cultured in complete medium and after 24 h, the cells were collected and PD-L1 expression was evaluated by flow cytometry and quantified as molecules of equivalent fluorochrome (MEF). ( B ) Cancer cells were treated with increasing concentrations of the indicated drugs for 72 h. IC 50 values ± SD were calculated using <t>GraphPad</t> Prism 6.00 software. ( C ) NSCLC cells were treated with gemcitabine, cisplatin, paclitaxel, vinorelbine, or pemetrexed at IC 50 concentrations. After 3 days, PD-L1 levels were analyzed by flow cytometry. ( D ) Confocal immunofluorescence analysis of PD-L1 expression in H322 cells maintained in the absence or in the presence of 100 nM pemetrexed for 72 h; green fluorescence indicates the positivity to PD-L1. The nuclei were stained with Draq5 (red fluorescence). Scale Bar: 10 μm. Data in ( A ) and ( B ) are mean values ± SD of three independent experiments. Results in ( C ) and ( D ) are representative of two independent experiments.
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    Overexpression of SUZ 12 mRNA in multiple HNSCC cohorts. The mRNA levels of SUZ 12 (log2‐transformed) were compared between HNSCC samples and normal counterparts in multiple patient cohorts. (A‐H) The original data were retrieved from Oncomine database and TCGA and then plotted using <t>GraphPad</t> Prism 6.0 software. Y ‐axis represents the median intensity, 25th and 75th percentile data. * P
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    GraphPad Prism Inc graphpad prism 6 04 software
    Overexpression of SUZ 12 mRNA in multiple HNSCC cohorts. The mRNA levels of SUZ 12 (log2‐transformed) were compared between HNSCC samples and normal counterparts in multiple patient cohorts. (A‐H) The original data were retrieved from Oncomine database and TCGA and then plotted using <t>GraphPad</t> Prism 6.0 software. Y ‐axis represents the median intensity, 25th and 75th percentile data. * P
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    GraphPad Prism Inc software package graphpad prism 6
    The lengths of the majority of D. chrysanthum pollen tubes present in compatible and incompatible styles at different times after pollination. Pollen tubes were measured at 12, 24, 48, 72, and 96 h after pollination. Each point is a mean value based on measurements of tubes in three styles, and the error bars represent ± the standard error of the mean. P -values were calculated by two-way repeated measures analysis of variance (ANOVA) using Sidak’s post hoc test in <t>GraphPad</t> <t>Prism</t> 6 ( ∗∗∗ P
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    Image Search Results


    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Statistic significance was tested using t -test (two-tailed P -value) using the GraphPad Prism 6.0 software.

    Techniques: Activity Assay, Software, Two Tailed Test, Produced, Concentration Assay

    Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Statistic significance was tested using t -test (two-tailed P -value) using the GraphPad Prism 6.0 software.

    Techniques: Activity Assay

    MTH1 deficiency sensitizes cells to Temozolomide. Cell viability of U251 (MTH1 proficient) and U251-MTH1 (MTH1 deficient) was measured using resazurin after 96 h of treatment with Temozolomide and Lomeguatrib. ( A ) EC 50 values for Temozolomide was determined to 62±4 μM for MTH1 proficient and 17±4 μM for MTH1 deficient cells with active MGMT, respectively. ( B ) EC 50 values for Temozolomide were 79±2 μM for MTH1 proficient and 11±2 μM for MTH1 deficient cells when MGMT is inhibited with 12.5 μM Lomeguatrib. MTH1 deficiency causes a 3.6-fold sensitization with active MGMT, and increases to 6.6-fold upon MGMT inhibition. Figures show representative experiments out of two independent experiments per treatment with data points in duplicate. Caspase 3/7 activity, detecting early apoptosis, was assayed using U251 and U251-MTH1 cells after treatment with Temozolomide ranging from 0 to 100 μM for 48 h ( C ) or with both Temozolomide and Lomeguatrib (12.5 μM) ( D ). MTH1 deficient cells show increased caspase 3/7 activity at 50 and 100 μM Temozolomide when MGMT is inhibited with 12.5 μM Lomeguatrib. Shown are representative experiments out of three independent experiments with data points in duplicate. Data are presented as average ± SEM. Statistical significant differences were determined using multiple comparison and Two way Anova in GraphPad Prism 6.0, P ≤ 0.01 is indicated by**.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: MTH1 deficiency sensitizes cells to Temozolomide. Cell viability of U251 (MTH1 proficient) and U251-MTH1 (MTH1 deficient) was measured using resazurin after 96 h of treatment with Temozolomide and Lomeguatrib. ( A ) EC 50 values for Temozolomide was determined to 62±4 μM for MTH1 proficient and 17±4 μM for MTH1 deficient cells with active MGMT, respectively. ( B ) EC 50 values for Temozolomide were 79±2 μM for MTH1 proficient and 11±2 μM for MTH1 deficient cells when MGMT is inhibited with 12.5 μM Lomeguatrib. MTH1 deficiency causes a 3.6-fold sensitization with active MGMT, and increases to 6.6-fold upon MGMT inhibition. Figures show representative experiments out of two independent experiments per treatment with data points in duplicate. Caspase 3/7 activity, detecting early apoptosis, was assayed using U251 and U251-MTH1 cells after treatment with Temozolomide ranging from 0 to 100 μM for 48 h ( C ) or with both Temozolomide and Lomeguatrib (12.5 μM) ( D ). MTH1 deficient cells show increased caspase 3/7 activity at 50 and 100 μM Temozolomide when MGMT is inhibited with 12.5 μM Lomeguatrib. Shown are representative experiments out of three independent experiments with data points in duplicate. Data are presented as average ± SEM. Statistical significant differences were determined using multiple comparison and Two way Anova in GraphPad Prism 6.0, P ≤ 0.01 is indicated by**.

    Article Snippet: Statistic significance was tested using t -test (two-tailed P -value) using the GraphPad Prism 6.0 software.

    Techniques: Inhibition, Activity Assay

    Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Article Snippet: Statistic significance was tested using t -test (two-tailed P -value) using the GraphPad Prism 6.0 software.

    Techniques: Incubation, Software, Injection, Fluorescence In Situ Hybridization, Inhibition, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    CSFV infection in vivo causes ER stress and differentially activates UPR pathways in infected organs. About 100 mg immune tissues (A–D) or non-immune tissues (E–J) as indicated from the experimental pigs were collected at 7 dpi for preparation of protein samples as described in “Materials and Methods,” samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against p-eIF2α, total eIF2α, ATF-4, CHOP, cleaved ATF-6, p-IRE1, GRP78 and tubulin. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses were performed with GraphPad Prism 6. All the data shown are expressed as mean ± SD values of three independent experiments. Multiple t tests: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: CSFV infection in vivo causes ER stress and differentially activates UPR pathways in infected organs. About 100 mg immune tissues (A–D) or non-immune tissues (E–J) as indicated from the experimental pigs were collected at 7 dpi for preparation of protein samples as described in “Materials and Methods,” samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against p-eIF2α, total eIF2α, ATF-4, CHOP, cleaved ATF-6, p-IRE1, GRP78 and tubulin. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses were performed with GraphPad Prism 6. All the data shown are expressed as mean ± SD values of three independent experiments. Multiple t tests: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Infection, In Vivo, BIA-KA, Western Blot, Software, Protein Quantitation

    CSFV infection in vivo differentially induces autophagy in infected organs. 100 mg immune tissues (A–D) or non-immune tissues (E–J) as indicated from the experimental pigs were collected at 7 dpi for preparation of protein samples as described in the “materials and methods”, samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against LC3B, p62, Beclin1, ATG5 and tubulin. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses were performed with GraphPad Prism 6. All the data shown are expressed as mean ± SD values of three independent experiments. Multiple t tests: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: CSFV infection in vivo differentially induces autophagy in infected organs. 100 mg immune tissues (A–D) or non-immune tissues (E–J) as indicated from the experimental pigs were collected at 7 dpi for preparation of protein samples as described in the “materials and methods”, samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against LC3B, p62, Beclin1, ATG5 and tubulin. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses were performed with GraphPad Prism 6. All the data shown are expressed as mean ± SD values of three independent experiments. Multiple t tests: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Infection, In Vivo, BIA-KA, Western Blot, Software, Protein Quantitation

    Autophagy-regulated CSFV replication in cultured PK-15 and 3D4/2 cells can be mediated by ER stress. (A) The cell viability of PK-15 and 3D4/2 (B) cells were measured by the CCK-8 assays after treatments with the indicated concentrations of TG (12.5, 25 and 50 nM), 4-PBA (125, 250 and 500 μM) and TUDCA (50, 100 and 200 μM). Cells pretreated with only DMSO (0.1%) or sterile ultrapure water were regarded as the control group. After further incubation for the indicated time as described in the “Materials and Methods”, cells were further incubated with 10% (v/v) CCK-8 diluted in fresh medium for 1 h at 37°C, followed by measurement of the absorbance at 450 nm with a Ledetect 96 microplate reader. Each sample was assayed in triplicate and the data represent the mean ± SD of two independent experiments. For virus titration assays, PK-15 (C) and 3D4/2 (D) cells cultured in 6-well microplates were pretreated with the indicated concentrations of TG, 4-PBA, RAPA and 3-MA for the indicated time, followed by infection with 0.5 MOI of CSFV. Twenty-four hours post-infection, the cells and supernatants of each treatment were collected for virus titration determined with TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody and Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) secondary antibody. Virus titers were calculated according to Reed-Muench method and expressed as Log 10 (TCID 50 /mL). Each sample was assayed in four replicates and the data represent the mean ± SD of two independent experiments. To investigate the effect of ER stress-mediated autophagy on CSFV replication in vitro , PK-15 (E) and 3D4/2 (F) cells cultured in 6-well plates were pretreated with 50 nM TG or 200 μM TUDCA for 6 h, and then subject to treatment with 100 nM RAPA for 1 h or 5 mM 3-MA for 4 h prior to CSFV infection. After a 1.5 h of incubation, the cells were further cultured in maintenance media in the presence (TUDCA, RAPA, 3-MA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only DMSO (0.1%) were regarded as the control group. The cells and supernatants of each treatment were collected for detecting the copies of NS5B genes and viral titers using real-time RT-PCR and TCID 50 assays, respectively. The results are expressed as mean ± SD values of two independent experiments. For all assays, generations of images for quantitation and all statistical analyses were performed with GraphPad Prism 6. All data presented were analyzed by one-way ANOVA tests: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: Autophagy-regulated CSFV replication in cultured PK-15 and 3D4/2 cells can be mediated by ER stress. (A) The cell viability of PK-15 and 3D4/2 (B) cells were measured by the CCK-8 assays after treatments with the indicated concentrations of TG (12.5, 25 and 50 nM), 4-PBA (125, 250 and 500 μM) and TUDCA (50, 100 and 200 μM). Cells pretreated with only DMSO (0.1%) or sterile ultrapure water were regarded as the control group. After further incubation for the indicated time as described in the “Materials and Methods”, cells were further incubated with 10% (v/v) CCK-8 diluted in fresh medium for 1 h at 37°C, followed by measurement of the absorbance at 450 nm with a Ledetect 96 microplate reader. Each sample was assayed in triplicate and the data represent the mean ± SD of two independent experiments. For virus titration assays, PK-15 (C) and 3D4/2 (D) cells cultured in 6-well microplates were pretreated with the indicated concentrations of TG, 4-PBA, RAPA and 3-MA for the indicated time, followed by infection with 0.5 MOI of CSFV. Twenty-four hours post-infection, the cells and supernatants of each treatment were collected for virus titration determined with TCID 50 assays through an indirect immunofluorescence assay using mouse anti-CSFV E2 antibody and Alexa Fluor 488-labeled goat anti-mouse IgG(H + L) secondary antibody. Virus titers were calculated according to Reed-Muench method and expressed as Log 10 (TCID 50 /mL). Each sample was assayed in four replicates and the data represent the mean ± SD of two independent experiments. To investigate the effect of ER stress-mediated autophagy on CSFV replication in vitro , PK-15 (E) and 3D4/2 (F) cells cultured in 6-well plates were pretreated with 50 nM TG or 200 μM TUDCA for 6 h, and then subject to treatment with 100 nM RAPA for 1 h or 5 mM 3-MA for 4 h prior to CSFV infection. After a 1.5 h of incubation, the cells were further cultured in maintenance media in the presence (TUDCA, RAPA, 3-MA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only DMSO (0.1%) were regarded as the control group. The cells and supernatants of each treatment were collected for detecting the copies of NS5B genes and viral titers using real-time RT-PCR and TCID 50 assays, respectively. The results are expressed as mean ± SD values of two independent experiments. For all assays, generations of images for quantitation and all statistical analyses were performed with GraphPad Prism 6. All data presented were analyzed by one-way ANOVA tests: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Cell Culture, CCK-8 Assay, Incubation, Titration, Infection, Immunofluorescence, Labeling, Endpoint Dilution Assay, In Vitro, Quantitative RT-PCR, Quantitation Assay

    Pharmacological regulation of ER stress changes expression of autophagic proteins in CSFV-infected PK-15 cells. PK-15 cells cultured in 6-well microplates were pretreated with ER stress agonist TG (12.5, 25 and 50 nM) (A) , ER stress inhibitors 4-PBA (125, 250 and 500 μM) (D) and TUDCA (50, 100 and 200 μM) (G) for 6 h, respectively, and then subject to CSFV infection. After a 1.5 h of incubation, the cells were further cultured in maintenance media in the presence (TUDCA, 4-PBA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only MDRV or mock infection or only drugs were regarded as the control group. Cells were collected for preparation of protein samples in accordance with description in the “materials and methods”. Samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against the indicated targets. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses (B,C,E,F,H,I) were performed with GraphPad Prism 6. All the pictures of western blot represent one of two independent experiments, and all the quantitative data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA test: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: Pharmacological regulation of ER stress changes expression of autophagic proteins in CSFV-infected PK-15 cells. PK-15 cells cultured in 6-well microplates were pretreated with ER stress agonist TG (12.5, 25 and 50 nM) (A) , ER stress inhibitors 4-PBA (125, 250 and 500 μM) (D) and TUDCA (50, 100 and 200 μM) (G) for 6 h, respectively, and then subject to CSFV infection. After a 1.5 h of incubation, the cells were further cultured in maintenance media in the presence (TUDCA, 4-PBA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only MDRV or mock infection or only drugs were regarded as the control group. Cells were collected for preparation of protein samples in accordance with description in the “materials and methods”. Samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against the indicated targets. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses (B,C,E,F,H,I) were performed with GraphPad Prism 6. All the pictures of western blot represent one of two independent experiments, and all the quantitative data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA test: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Expressing, Infection, Cell Culture, Incubation, BIA-KA, Western Blot, Software, Protein Quantitation

    Pharmacological regulation of ER stress alters expression autophagic proteins in CSFV-infected 3D4/2 cells. 3D4/2 cells were treated with the indicated concentrations of TG (A) , 4-PBA (D) and TUDCA (F) prior to viral infection as described in the legend of Figure 6 , and then collected for preparation of protein samples according to the description in the “Materials and Methods”. Samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against the indicated targets. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses (B,C,E,G) were performed with GraphPad Prism 6. All the pictures of western blot represent one of two independent experiments, and all the quantitative data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA test: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: Pharmacological regulation of ER stress alters expression autophagic proteins in CSFV-infected 3D4/2 cells. 3D4/2 cells were treated with the indicated concentrations of TG (A) , 4-PBA (D) and TUDCA (F) prior to viral infection as described in the legend of Figure 6 , and then collected for preparation of protein samples according to the description in the “Materials and Methods”. Samples were quantitated by BCA assays and then loaded for western blot analyses using specific antibody against the indicated targets. Tubulin was used as a loading control. Grayscale value of the bands were analyzed with ImageJ software, and generations of images for protein quantitation and all statistical analyses (B,C,E,G) were performed with GraphPad Prism 6. All the pictures of western blot represent one of two independent experiments, and all the quantitative data shown are expressed as mean ± SD values of two independent experiments. Two-way ANOVA test: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Expressing, Infection, BIA-KA, Western Blot, Software, Protein Quantitation

    Viral loads of CSFV vary with the degree of injury in infected organs. About 100 mg tissues as indicated from the experimental pigs were collected for extraction of total RNA, 1 μg total RNA was transcribed into cDNA as template for absolute quantification of CSFV NS5B gene copies through real-time RT-PCR. A recombinant plasmid pMD18-T- NS5B was used as standard samples. The standard curve was constructed using 10-fold serially diluted standard plasmids. A linear regression plot was generated using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, Calif). The slope, the Y-intercept and the R 2 value were determined (A) . The viral loads in terms of NS5B copies/g tissues from the experimental pigs (B–D) were determined using the linear regression plot. Each template was ran in triplicate.

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: Viral loads of CSFV vary with the degree of injury in infected organs. About 100 mg tissues as indicated from the experimental pigs were collected for extraction of total RNA, 1 μg total RNA was transcribed into cDNA as template for absolute quantification of CSFV NS5B gene copies through real-time RT-PCR. A recombinant plasmid pMD18-T- NS5B was used as standard samples. The standard curve was constructed using 10-fold serially diluted standard plasmids. A linear regression plot was generated using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, Calif). The slope, the Y-intercept and the R 2 value were determined (A) . The viral loads in terms of NS5B copies/g tissues from the experimental pigs (B–D) were determined using the linear regression plot. Each template was ran in triplicate.

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Infection, Quantitative RT-PCR, Recombinant, Plasmid Preparation, Construct, Generated, Software

    Treatment with ER stress regulators changes transcriptional levels of autophagic genes in CSFV-infected PK-15 and 3D4/2 cells. PK-15 (A,B) and 3D4/2 (C,D) cells cultured in 6-well plates were pretreated with 50 nM TG or 200 μM TUDCA or 500 μM 4-PBA for 6 h, and then subject to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured in maintenance media in the presence (TUDCA and 4-PBA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only DMSO (0.1%) were regarded as the control group. The CSFV group was only infected with 1 MOI of CSFV, and the MOCK group was only treated with the same amount of media. The cells of each treatment were collected for extraction of total RNA, and 1 μg total RNA from each sample was transcribed into cDNA as template for relative quantification of the indicated genes through real-time RT-PCR. Relative mRNA expression of the target genes were assessed using the 2 −ΔΔCT method and normalized to the GAPDH gene. Each template was ran in triplicate. The results are expressed as mean ± SD values of two independent experiments. Generations of images for quantitation and statistical analyses were performed with GraphPad Prism 6. All data presented were analyzed by two-way ANOV A tests: * p

    Journal: Frontiers in Microbiology

    Article Title: Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro

    doi: 10.3389/fmicb.2019.02545

    Figure Lengend Snippet: Treatment with ER stress regulators changes transcriptional levels of autophagic genes in CSFV-infected PK-15 and 3D4/2 cells. PK-15 (A,B) and 3D4/2 (C,D) cells cultured in 6-well plates were pretreated with 50 nM TG or 200 μM TUDCA or 500 μM 4-PBA for 6 h, and then subject to a 1.5 h of incubation with 1 MOI of CSFV. The cells were further cultured in maintenance media in the presence (TUDCA and 4-PBA) or absence (TG) of the drugs for 24 h. Additionally, cells pretreated with only DMSO (0.1%) were regarded as the control group. The CSFV group was only infected with 1 MOI of CSFV, and the MOCK group was only treated with the same amount of media. The cells of each treatment were collected for extraction of total RNA, and 1 μg total RNA from each sample was transcribed into cDNA as template for relative quantification of the indicated genes through real-time RT-PCR. Relative mRNA expression of the target genes were assessed using the 2 −ΔΔCT method and normalized to the GAPDH gene. Each template was ran in triplicate. The results are expressed as mean ± SD values of two independent experiments. Generations of images for quantitation and statistical analyses were performed with GraphPad Prism 6. All data presented were analyzed by two-way ANOV A tests: * p

    Article Snippet: Statistical Analysis The data in the study are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or two-way ANOVA using the GraphPad prism 6 software. p less than 0.05 were considered statistically significant.

    Techniques: Infection, Cell Culture, Incubation, Quantitative RT-PCR, Expressing, Quantitation Assay

    ATP-dependent transport of TCA (○), GCA (▲) and CA (□) in membrane vesicles (7.5 μg protein) expressing human, dog and cat BSEP/Bsep. Vesicles were incubated in 10 mMTris buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min (TCA) or 7.5 min (CA and GCA). ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Curves were fitted by using GraphPad Prism 6.01 software (San Diego, California, USA). Measurements were performed in duplicate in three independent experiments and are given in pmol/mg protein/min. Each value in the graph is the mean ± SD of three independent experiments.

    Journal: BMC Veterinary Research

    Article Title: The feline bile salt export pump: a structural and functional comparison with canine and human Bsep/BSEP

    doi: 10.1186/1746-6148-9-259

    Figure Lengend Snippet: ATP-dependent transport of TCA (○), GCA (▲) and CA (□) in membrane vesicles (7.5 μg protein) expressing human, dog and cat BSEP/Bsep. Vesicles were incubated in 10 mMTris buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min (TCA) or 7.5 min (CA and GCA). ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Curves were fitted by using GraphPad Prism 6.01 software (San Diego, California, USA). Measurements were performed in duplicate in three independent experiments and are given in pmol/mg protein/min. Each value in the graph is the mean ± SD of three independent experiments.

    Article Snippet: Concentration-dependent BSEP transport rates were fitted according to Michaelis-Menten enzyme kinetics and Km values were calculated by means of GraphPad Prism 6.01 software (San Diego, California, USA).

    Techniques: Expressing, Incubation, Software

    ATP-dependent transport of TCA, CA and GCA in membrane vesicles (7.5 μg protein) expressing BSEP/Bsep of human (▲), dogs (□), and cats (○). Vesicles were incubated in 10 mMTris Base buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min (TCA) or 7.5 min (CA and GCA). ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Curves were fitted by GraphPad Prism 6.01 software (San Diego, California, USA) and V max was calculated. Measurements were performed in duplicate in at least three independent experiments. The mean of the duplicates of each experiment was transformed as a relative activity to the V max (=100%). Each value in the graph is the mean ± SD of the relative activities of three independent experiments.

    Journal: BMC Veterinary Research

    Article Title: The feline bile salt export pump: a structural and functional comparison with canine and human Bsep/BSEP

    doi: 10.1186/1746-6148-9-259

    Figure Lengend Snippet: ATP-dependent transport of TCA, CA and GCA in membrane vesicles (7.5 μg protein) expressing BSEP/Bsep of human (▲), dogs (□), and cats (○). Vesicles were incubated in 10 mMTris Base buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min (TCA) or 7.5 min (CA and GCA). ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Curves were fitted by GraphPad Prism 6.01 software (San Diego, California, USA) and V max was calculated. Measurements were performed in duplicate in at least three independent experiments. The mean of the duplicates of each experiment was transformed as a relative activity to the V max (=100%). Each value in the graph is the mean ± SD of the relative activities of three independent experiments.

    Article Snippet: Concentration-dependent BSEP transport rates were fitted according to Michaelis-Menten enzyme kinetics and Km values were calculated by means of GraphPad Prism 6.01 software (San Diego, California, USA).

    Techniques: Expressing, Incubation, Software, Transformation Assay, Activity Assay

    Inhibition of cyclosporine A, troglitazone and ketoconazole on the uptake of 1 μM [ 3 H]TCA in membrane vesicles (7.5 μg protein) expressing human (▲), dog (□) and cat (○) BSEP/Bsep. Vesicles were incubated in 10 mMTris Base buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min. ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Inhibition curves were fitted by using GraphPad Prism 6.01 software (San Diego, California, USA) and values are expressed as mean ± SD of percentage uptake of fourindependent experiments.

    Journal: BMC Veterinary Research

    Article Title: The feline bile salt export pump: a structural and functional comparison with canine and human Bsep/BSEP

    doi: 10.1186/1746-6148-9-259

    Figure Lengend Snippet: Inhibition of cyclosporine A, troglitazone and ketoconazole on the uptake of 1 μM [ 3 H]TCA in membrane vesicles (7.5 μg protein) expressing human (▲), dog (□) and cat (○) BSEP/Bsep. Vesicles were incubated in 10 mMTris Base buffer (pH 7.4) containing 250 mM Sucrose, 10 mM MgCl 2 and 4 mM ATP or AMP, at 37°C for 5 min. ATP-dependent transport was calculated by subtracting transport in presence of AMP from that in presence of ATP. Inhibition curves were fitted by using GraphPad Prism 6.01 software (San Diego, California, USA) and values are expressed as mean ± SD of percentage uptake of fourindependent experiments.

    Article Snippet: Concentration-dependent BSEP transport rates were fitted according to Michaelis-Menten enzyme kinetics and Km values were calculated by means of GraphPad Prism 6.01 software (San Diego, California, USA).

    Techniques: Inhibition, Expressing, Incubation, Software

    Differentiation of primary rat oligodendrocyte progenitor cells (OPCs) using distinct substrates and conditions. ( A ) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for MBP (in red, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. ( B ) The percentage of MBP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent mean ± SEM of at least five independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. ( C ) Quantification of CTCF of MBP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t -test. ( D,F ) Measurement of MBP signal area of primary rat oligodendrocytes cultured with differentiation medium for 3 days ( D ) or 5 days ( F ) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ( E,G ) Mean fluorescence intensity (MFI) of MBP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days ( E ) or for 5 days ( G ). Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t -test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s non-significant, * p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Differentiation of primary rat oligodendrocyte progenitor cells (OPCs) using distinct substrates and conditions. ( A ) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for MBP (in red, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. ( B ) The percentage of MBP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent mean ± SEM of at least five independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. ( C ) Quantification of CTCF of MBP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t -test. ( D,F ) Measurement of MBP signal area of primary rat oligodendrocytes cultured with differentiation medium for 3 days ( D ) or 5 days ( F ) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ( E,G ) Mean fluorescence intensity (MFI) of MBP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days ( E ) or for 5 days ( G ). Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t -test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s non-significant, * p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Immunofluorescence, Microscopy, Staining, Cell Culture, Fluorescence, Software

    Effect of substrate stiffness, merosin and actomyosin contractility inhibition on morphological features of oligodendrocytes during early-stage differentiation. ( A ) Representative fluorescence microscopy images of OPCs cultured for 24 h in differentiation medium on 6.5 kPa PAHs functionalised with PDLMN in absence (left) or presence (right) of 15 μM of non-muscle myosin-II (NMM-II) inhibitor blebbistatin. Cells were immunostained for the oligodendrocyte lineage marker Olig2 (nuclear staining, in red) and for alpha-tubulin to stain the processes (green) and nuclei were counterstained with DAPI (blue). Scale bar corresponds to 50 μm. The area of oligodendrocyte processes of Olig2-positive cells — e.g.: cells highlighted by pink arrowheads in ( A ) — maintained on 6.5 kPa PAHs ( B ) or on TCPs ( C ) were normalised to the conditions PAHs PDL ( B ) or TCPs PDL ( C ). Data in graphs represent n = 30 cells obtained from three independent experiments. Statistical analysis was performed by t -test using the software GraphPad Prism 6. Statistical significance between conditions under comparison was represented using connectors (n.s.: non-significant, * p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Effect of substrate stiffness, merosin and actomyosin contractility inhibition on morphological features of oligodendrocytes during early-stage differentiation. ( A ) Representative fluorescence microscopy images of OPCs cultured for 24 h in differentiation medium on 6.5 kPa PAHs functionalised with PDLMN in absence (left) or presence (right) of 15 μM of non-muscle myosin-II (NMM-II) inhibitor blebbistatin. Cells were immunostained for the oligodendrocyte lineage marker Olig2 (nuclear staining, in red) and for alpha-tubulin to stain the processes (green) and nuclei were counterstained with DAPI (blue). Scale bar corresponds to 50 μm. The area of oligodendrocyte processes of Olig2-positive cells — e.g.: cells highlighted by pink arrowheads in ( A ) — maintained on 6.5 kPa PAHs ( B ) or on TCPs ( C ) were normalised to the conditions PAHs PDL ( B ) or TCPs PDL ( C ). Data in graphs represent n = 30 cells obtained from three independent experiments. Statistical analysis was performed by t -test using the software GraphPad Prism 6. Statistical significance between conditions under comparison was represented using connectors (n.s.: non-significant, * p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Inhibition, Fluorescence, Microscopy, Cell Culture, Marker, Staining, Software

    Cell morphology assessment of CG-4 cells by fractal dimension analysis. ( A ) Representative fluorescence microscopy images of oligodendroglial CG-4 cells plated on glass coverslips or 6.5 kPa polyacrylamide hydrogels (PAHs) functionalised with fibronectin (FN) or poly-D-lysine and merosin (PDLMN) and maintained in proliferation medium for 2 days. Scale bar corresponds to 50 μm. The D values (fractal dimension) are shown in ( B,C ). Values in ( B ) represent at least n = 13 cells analysed from three independent experiments and in ( C ) are depicted representative images of cells analysed in ( B ). Statistical analysis was performed by t-test using the software GraphPad Prism 6. Statistical comparisons were represented using connectors (n.s.: non-significant, *** p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Cell morphology assessment of CG-4 cells by fractal dimension analysis. ( A ) Representative fluorescence microscopy images of oligodendroglial CG-4 cells plated on glass coverslips or 6.5 kPa polyacrylamide hydrogels (PAHs) functionalised with fibronectin (FN) or poly-D-lysine and merosin (PDLMN) and maintained in proliferation medium for 2 days. Scale bar corresponds to 50 μm. The D values (fractal dimension) are shown in ( B,C ). Values in ( B ) represent at least n = 13 cells analysed from three independent experiments and in ( C ) are depicted representative images of cells analysed in ( B ). Statistical analysis was performed by t-test using the software GraphPad Prism 6. Statistical comparisons were represented using connectors (n.s.: non-significant, *** p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Fluorescence, Microscopy, Software

    Morphological characterization of oligodendrocytes differentiated using distinct substrates and conditions. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using distinct combinations of substrates (TCPs or 6.5 kPa PAHs) coated/functionalised with PDL or PDLMN, as indicated. Immunostaining was performed using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( C ) Quantification of the percentage of cells bearing a non-membranous, membranous, or myelin sheet distribution of MBP, as depicted in the representative images in ( B ) — left, centre and right panels, respectively. The graph represents data from six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test using the software GraphPad Prism 6. Statistically significant differences within cellular categories between substrates were represented (*, # p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Morphological characterization of oligodendrocytes differentiated using distinct substrates and conditions. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using distinct combinations of substrates (TCPs or 6.5 kPa PAHs) coated/functionalised with PDL or PDLMN, as indicated. Immunostaining was performed using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( C ) Quantification of the percentage of cells bearing a non-membranous, membranous, or myelin sheet distribution of MBP, as depicted in the representative images in ( B ) — left, centre and right panels, respectively. The graph represents data from six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test using the software GraphPad Prism 6. Statistically significant differences within cellular categories between substrates were represented (*, # p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Fluorescence, Microscopy, Cell Culture, Immunostaining, Software

    Maturation of primary rat OPCs using distinct substrates and conditions. ( A ) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for PLP (in green, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM, respectively). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. ( B ) The percentage of PLP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. ( C ) Quantification of CTCF for PLP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. ( D,F ) Measurement of the PLP signal area of primary rat oligodendrocytes cultured with DM for 3 days ( D ) or 5 days ( F ) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ( E,G ) Mean fluorescence intensity (MFI) of PLP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days ( E ) or for 5 days ( G ). Data represent mean ± SEM of CTCF of at least 3 independent experiments. Statistical analysis was performed by t -test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s.: non-significant, * p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Maturation of primary rat OPCs using distinct substrates and conditions. ( A ) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for PLP (in green, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM, respectively). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. ( B ) The percentage of PLP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. ( C ) Quantification of CTCF for PLP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. ( D,F ) Measurement of the PLP signal area of primary rat oligodendrocytes cultured with DM for 3 days ( D ) or 5 days ( F ) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ( E,G ) Mean fluorescence intensity (MFI) of PLP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days ( E ) or for 5 days ( G ). Data represent mean ± SEM of CTCF of at least 3 independent experiments. Statistical analysis was performed by t -test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s.: non-significant, * p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Immunofluorescence, Microscopy, Staining, Plasmid Purification, Cell Culture, Fluorescence, Software

    Modulation of oligodendrocyte differentiation and MBP levels by substrate stiffness within a narrow, brain-compliant range. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with 7 PDL or PDLMN, as indicated, using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( B ) Percentage of MBP-positive cells on the distinct substrates and ( C ) quantification of CTCF for MBP of oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). ( D ) Measurement of MBP signal area and ( E ) mean fluorescence intensity (MFI) of MBP signal (six fields per experiment from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test [in ( B )] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (* p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Modulation of oligodendrocyte differentiation and MBP levels by substrate stiffness within a narrow, brain-compliant range. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with 7 PDL or PDLMN, as indicated, using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( B ) Percentage of MBP-positive cells on the distinct substrates and ( C ) quantification of CTCF for MBP of oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). ( D ) Measurement of MBP signal area and ( E ) mean fluorescence intensity (MFI) of MBP signal (six fields per experiment from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test [in ( B )] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (* p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Fluorescence, Microscopy, Cell Culture, Software

    Modulation of oligodendrocyte maturation and PLP levels by substrate stiffness within a narrow, brain-compliant range. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with PDL +/− MN, as indicated, using an anti-PLP antibody (green) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( B ) Percentage of the PLP-positive cells on distinct substrates and ( C ) quantification of CTCF for PLP of primary oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). ( D ) Measurement of the PLP signal area and ( E ) mean fluorescence intensity (MFI) of the PLP signal (six fields from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison or Kuskal-Wallis test [in ( B )] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (* p

    Journal: Scientific Reports

    Article Title: Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    doi: 10.1038/srep21563

    Figure Lengend Snippet: Modulation of oligodendrocyte maturation and PLP levels by substrate stiffness within a narrow, brain-compliant range. ( A ) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with PDL +/− MN, as indicated, using an anti-PLP antibody (green) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. ( B ) Percentage of the PLP-positive cells on distinct substrates and ( C ) quantification of CTCF for PLP of primary oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). ( D ) Measurement of the PLP signal area and ( E ) mean fluorescence intensity (MFI) of the PLP signal (six fields from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison or Kuskal-Wallis test [in ( B )] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (* p

    Article Snippet: Analysis was done using the software GraphPad Prism 6.

    Techniques: Plasmid Purification, Fluorescence, Microscopy, Cell Culture, Software

    P2X4 and P2X7 expressions in HCC. The expression level ( y -axis) and receptor types ( x -axis) have been plotted for the 20 samples of HCC tissue for P2X4 and P2X7 separately. More than 200% expression level of P2X4 and P2X7 was observed in the HCC as compared with control tissue samples. ANOVA analysis was performed in GraphPad Prism 6.0. All values are expressed in mean SEM. *** P value

    Journal: Purinergic Signalling

    Article Title: Role of purinergic receptors in hepatobiliary carcinoma in Pakistani population: an approach towards proinflammatory role of P2X4 and P2X7 receptors

    doi: 10.1007/s11302-019-09675-0

    Figure Lengend Snippet: P2X4 and P2X7 expressions in HCC. The expression level ( y -axis) and receptor types ( x -axis) have been plotted for the 20 samples of HCC tissue for P2X4 and P2X7 separately. More than 200% expression level of P2X4 and P2X7 was observed in the HCC as compared with control tissue samples. ANOVA analysis was performed in GraphPad Prism 6.0. All values are expressed in mean SEM. *** P value

    Article Snippet: The statistical software GraphPad Prism 6.0 was used for the statistical analysis.

    Techniques: Expressing

    P2X4 and P2X7 expressions in adenocarcinoma. The expression level ( y -axis) and receptor types ( x -axis) have been plotted for the 10 samples of ADC for P2X4 and P2X7 receptors separately. As compared with the control samples, P2X4 receptor expression was significantly high with more than 200% increase while the expression of P2X7 receptor was about 100% higher than that of the control samples. ANOVA analysis was performed in GraphPad Prism 6.0. All values are expressed in mean SEM. *** P value

    Journal: Purinergic Signalling

    Article Title: Role of purinergic receptors in hepatobiliary carcinoma in Pakistani population: an approach towards proinflammatory role of P2X4 and P2X7 receptors

    doi: 10.1007/s11302-019-09675-0

    Figure Lengend Snippet: P2X4 and P2X7 expressions in adenocarcinoma. The expression level ( y -axis) and receptor types ( x -axis) have been plotted for the 10 samples of ADC for P2X4 and P2X7 receptors separately. As compared with the control samples, P2X4 receptor expression was significantly high with more than 200% increase while the expression of P2X7 receptor was about 100% higher than that of the control samples. ANOVA analysis was performed in GraphPad Prism 6.0. All values are expressed in mean SEM. *** P value

    Article Snippet: The statistical software GraphPad Prism 6.0 was used for the statistical analysis.

    Techniques: Expressing

    In vivo topical pre- or post-application of BAY 11-7082 prevents acidic bile-induced cell proliferation markerKi67 in 10-day exposed murine HM. ( A ) (a) IHC analysis for Ki67 (from left to right): control untreated HM , revealing weak cytoplasmic or nuclear staining in few basal cells; saline-DMSO treated HM , demonstrating sporadic and weak cytoplasmic or nuclear staining in cells of the basal layer; acidic bile-treated HM , revealing intense nuclear and cytoplasmic staining of cells of basal and parabasal layers; pre-application inhibitor (BAY 11 7082) + acidic bile-treated HM and acidic bile + post-application inhibitor (BAY 11 7082)-treated HM, producing weak nuclear or cytoplasmic staining in cells of the basal layer. (b) Image analysis algorithm (s) (red: nuclear positive staining of Ki67; orange: intense positive cytoplasmic staining of Ki67; yellow: weak cytoplasmic staining of Ki67; blue: negative Ki67 staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm (s) illustrating the mucosal and cellular Ki67 staining compartments]. ( B ) Graphs depict 10-day short-term exposure of HM to acidic bile (pH 3.0) inducing significantly higher positive nuclear Ki67 levels compared to saline-treated HM or untreated control. Topical pre- or post-application of BAY 11-7082 significantly decreases nuclear Ki67 levels in acidic bile (pH 3.0) HM ( p values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total-positive Ki67 staining).

    Journal: Oncotarget

    Article Title: The temporal effects of topical NF-κB inhibition, in the in vivo prevention of bile-related oncogenic mRNA and miRNA phenotypes in murine hypopharyngeal mucosa: a preclinical model

    doi: 10.18632/oncotarget.27706

    Figure Lengend Snippet: In vivo topical pre- or post-application of BAY 11-7082 prevents acidic bile-induced cell proliferation markerKi67 in 10-day exposed murine HM. ( A ) (a) IHC analysis for Ki67 (from left to right): control untreated HM , revealing weak cytoplasmic or nuclear staining in few basal cells; saline-DMSO treated HM , demonstrating sporadic and weak cytoplasmic or nuclear staining in cells of the basal layer; acidic bile-treated HM , revealing intense nuclear and cytoplasmic staining of cells of basal and parabasal layers; pre-application inhibitor (BAY 11 7082) + acidic bile-treated HM and acidic bile + post-application inhibitor (BAY 11 7082)-treated HM, producing weak nuclear or cytoplasmic staining in cells of the basal layer. (b) Image analysis algorithm (s) (red: nuclear positive staining of Ki67; orange: intense positive cytoplasmic staining of Ki67; yellow: weak cytoplasmic staining of Ki67; blue: negative Ki67 staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm (s) illustrating the mucosal and cellular Ki67 staining compartments]. ( B ) Graphs depict 10-day short-term exposure of HM to acidic bile (pH 3.0) inducing significantly higher positive nuclear Ki67 levels compared to saline-treated HM or untreated control. Topical pre- or post-application of BAY 11-7082 significantly decreases nuclear Ki67 levels in acidic bile (pH 3.0) HM ( p values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total-positive Ki67 staining).

    Article Snippet: Statistical analysis GraphPad Prism 6 software and one-way analysis of variance (ANOVA) (Friedman test and Dunn’s multiple-analysis test were used to determine significance defined as p values < 0.05).

    Techniques: In Vivo, Immunohistochemistry, Staining, Software, Generated

    In vivo topical pre- or post-application of BAY 11-7082 inhibits the acidic bile-induced NF- κ B activation in 10-days exposed murine HM. ( A ) (a) IHC analysis for p-NF- κ B (p65 S536) (from left to right): control untreated HM, revealing cytoplasmic staining; saline-DMSO treated HM, demonstrating sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile-treated HM, producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; pre-application inhibitor (BAY 11 7082) + acidic bile-treated HM, demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers; acidic bile + post-application inhibitor (BAY 11 7082)-treated HM, demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and a weak nuclear or cytoplasmic staining of few cells in suprabasal layers; (b) Image analysis algorithm (s) (red: positive nuclear staining of p-NF- κ B; orange: intense positive cytoplasmic staining of p-NF- κ B; yellow: weak cytoplasmic staining of p-NF- κ B; blue: negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm (s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. ( B ) Nuclear positivity of p-NF- κ B (p65 S536) in murine HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total positive p-p65 staining). Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-DMSO treated HM or untreated control. Topical pre- or post-application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( p values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total positive p-p65 staining).

    Journal: Oncotarget

    Article Title: The temporal effects of topical NF-κB inhibition, in the in vivo prevention of bile-related oncogenic mRNA and miRNA phenotypes in murine hypopharyngeal mucosa: a preclinical model

    doi: 10.18632/oncotarget.27706

    Figure Lengend Snippet: In vivo topical pre- or post-application of BAY 11-7082 inhibits the acidic bile-induced NF- κ B activation in 10-days exposed murine HM. ( A ) (a) IHC analysis for p-NF- κ B (p65 S536) (from left to right): control untreated HM, revealing cytoplasmic staining; saline-DMSO treated HM, demonstrating sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile-treated HM, producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; pre-application inhibitor (BAY 11 7082) + acidic bile-treated HM, demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers; acidic bile + post-application inhibitor (BAY 11 7082)-treated HM, demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and a weak nuclear or cytoplasmic staining of few cells in suprabasal layers; (b) Image analysis algorithm (s) (red: positive nuclear staining of p-NF- κ B; orange: intense positive cytoplasmic staining of p-NF- κ B; yellow: weak cytoplasmic staining of p-NF- κ B; blue: negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm (s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. ( B ) Nuclear positivity of p-NF- κ B (p65 S536) in murine HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total positive p-p65 staining). Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-DMSO treated HM or untreated control. Topical pre- or post-application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( p values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity = nuclear-positive/total positive p-p65 staining).

    Article Snippet: Statistical analysis GraphPad Prism 6 software and one-way analysis of variance (ANOVA) (Friedman test and Dunn’s multiple-analysis test were used to determine significance defined as p values < 0.05).

    Techniques: In Vivo, Activation Assay, Immunohistochemistry, Staining, Software, Generated

    Quantitative northern blot analysis of miRNA-like candidates ( a ) miRCat-2 and ( b ) Folded-2. Individual lanes were loaded with 4 μg sRNA which were blotted and onto membranes and hybridised at 37 °C overnight with. P 32 labelled oligonucleotides designed to detect miRNA-like candidate species. A. fumigatus U1-1 small nuclear RNA was used as a loading control throughout and the miRNA bands were normalised using the integrity of the U1-1 bands. Relative expression levels were plotted using the GraphPad Prism 6.0 programme and P values were estimated using unpaired t -test with Welch’s correction. Error bars were calculated using the standard error values. P values less than 0.05 were accepted as statistically significantly different and are shown with an asterisk (** indicates P ≤ 0.01; n = 3)

    Journal: BMC Genomics

    Article Title: Profile and functional analysis of small RNAs derived from Aspergillus fumigatus infected with double-stranded RNA mycoviruses

    doi: 10.1186/s12864-017-3773-8

    Figure Lengend Snippet: Quantitative northern blot analysis of miRNA-like candidates ( a ) miRCat-2 and ( b ) Folded-2. Individual lanes were loaded with 4 μg sRNA which were blotted and onto membranes and hybridised at 37 °C overnight with. P 32 labelled oligonucleotides designed to detect miRNA-like candidate species. A. fumigatus U1-1 small nuclear RNA was used as a loading control throughout and the miRNA bands were normalised using the integrity of the U1-1 bands. Relative expression levels were plotted using the GraphPad Prism 6.0 programme and P values were estimated using unpaired t -test with Welch’s correction. Error bars were calculated using the standard error values. P values less than 0.05 were accepted as statistically significantly different and are shown with an asterisk (** indicates P ≤ 0.01; n = 3)

    Article Snippet: Statistical analysis GraphPad Prism 6.0 software was used to analyse the qPCR results and P values were estimated using unpaired t -test with Welch’s correction.

    Techniques: Northern Blot, Expressing

    ( a ) Flow cytometry analysis of MDA-MB-231 and MCF-10A cells under action of B. salicifolius essential oil; ( b ) Bar diagram demonstrates that there is no difference between control cells and treated cells at the concentration of essential oil determined by cytotoxic activity assays for both cells lines. Ctrl: control; Bs: treatment with B. salicifolius essential oil. Data are expressed as mean ± standard deviation for experiments carried out in triplicate. Comparison between bars was performed by two-way ANOVA test followed by Sidak’s test using GraphPad Prism 6.01.

    Journal: International Journal of Molecular Sciences

    Article Title: Chemical Composition and Bioactivity of Essential Oil from Blepharocalyx salicifolius

    doi: 10.3390/ijms19010033

    Figure Lengend Snippet: ( a ) Flow cytometry analysis of MDA-MB-231 and MCF-10A cells under action of B. salicifolius essential oil; ( b ) Bar diagram demonstrates that there is no difference between control cells and treated cells at the concentration of essential oil determined by cytotoxic activity assays for both cells lines. Ctrl: control; Bs: treatment with B. salicifolius essential oil. Data are expressed as mean ± standard deviation for experiments carried out in triplicate. Comparison between bars was performed by two-way ANOVA test followed by Sidak’s test using GraphPad Prism 6.01.

    Article Snippet: For flow cytometry analysis, two-way ANOVA followed by Sidak’s test was performed using the software GraphPad Prism 6.01.

    Techniques: Flow Cytometry, Cytometry, Multiple Displacement Amplification, Concentration Assay, Activity Assay, Standard Deviation

    Modulation of programmed cell death ligand-1 (PD-L1) levels by chemotherapeutic drugs in non-small-cell lung cancer (NSCLC) cell lines. ( A ) A549, Calu-6, H292, and H322 cells were cultured in complete medium and after 24 h, the cells were collected and PD-L1 expression was evaluated by flow cytometry and quantified as molecules of equivalent fluorochrome (MEF). ( B ) Cancer cells were treated with increasing concentrations of the indicated drugs for 72 h. IC 50 values ± SD were calculated using GraphPad Prism 6.00 software. ( C ) NSCLC cells were treated with gemcitabine, cisplatin, paclitaxel, vinorelbine, or pemetrexed at IC 50 concentrations. After 3 days, PD-L1 levels were analyzed by flow cytometry. ( D ) Confocal immunofluorescence analysis of PD-L1 expression in H322 cells maintained in the absence or in the presence of 100 nM pemetrexed for 72 h; green fluorescence indicates the positivity to PD-L1. The nuclei were stained with Draq5 (red fluorescence). Scale Bar: 10 μm. Data in ( A ) and ( B ) are mean values ± SD of three independent experiments. Results in ( C ) and ( D ) are representative of two independent experiments.

    Journal: Cancers

    Article Title: Pemetrexed Enhances Membrane PD-L1 Expression and Potentiates T Cell-Mediated Cytotoxicity by Anti-PD-L1 Antibody Therapy in Non-Small-Cell Lung Cancer

    doi: 10.3390/cancers12030666

    Figure Lengend Snippet: Modulation of programmed cell death ligand-1 (PD-L1) levels by chemotherapeutic drugs in non-small-cell lung cancer (NSCLC) cell lines. ( A ) A549, Calu-6, H292, and H322 cells were cultured in complete medium and after 24 h, the cells were collected and PD-L1 expression was evaluated by flow cytometry and quantified as molecules of equivalent fluorochrome (MEF). ( B ) Cancer cells were treated with increasing concentrations of the indicated drugs for 72 h. IC 50 values ± SD were calculated using GraphPad Prism 6.00 software. ( C ) NSCLC cells were treated with gemcitabine, cisplatin, paclitaxel, vinorelbine, or pemetrexed at IC 50 concentrations. After 3 days, PD-L1 levels were analyzed by flow cytometry. ( D ) Confocal immunofluorescence analysis of PD-L1 expression in H322 cells maintained in the absence or in the presence of 100 nM pemetrexed for 72 h; green fluorescence indicates the positivity to PD-L1. The nuclei were stained with Draq5 (red fluorescence). Scale Bar: 10 μm. Data in ( A ) and ( B ) are mean values ± SD of three independent experiments. Results in ( C ) and ( D ) are representative of two independent experiments.

    Article Snippet: Statistical Analysis Statistical analyses were carried out using the GraphPad Prism 6.00 software.

    Techniques: Cell Culture, Expressing, Flow Cytometry, Software, Immunofluorescence, Fluorescence, Staining

    Overexpression of SUZ 12 mRNA in multiple HNSCC cohorts. The mRNA levels of SUZ 12 (log2‐transformed) were compared between HNSCC samples and normal counterparts in multiple patient cohorts. (A‐H) The original data were retrieved from Oncomine database and TCGA and then plotted using GraphPad Prism 6.0 software. Y ‐axis represents the median intensity, 25th and 75th percentile data. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma, et al. SUZ12 is a novel putative oncogene promoting tumorigenesis in head and neck squamous cell carcinoma

    doi: 10.1111/jcmm.13638

    Figure Lengend Snippet: Overexpression of SUZ 12 mRNA in multiple HNSCC cohorts. The mRNA levels of SUZ 12 (log2‐transformed) were compared between HNSCC samples and normal counterparts in multiple patient cohorts. (A‐H) The original data were retrieved from Oncomine database and TCGA and then plotted using GraphPad Prism 6.0 software. Y ‐axis represents the median intensity, 25th and 75th percentile data. * P

    Article Snippet: All statistical analyses were performed with GraphPad Prism 6 (GraphPad) or SPSS 21.0 software (IBM).

    Techniques: Over Expression, Transformation Assay, Software

    The lengths of the majority of D. chrysanthum pollen tubes present in compatible and incompatible styles at different times after pollination. Pollen tubes were measured at 12, 24, 48, 72, and 96 h after pollination. Each point is a mean value based on measurements of tubes in three styles, and the error bars represent ± the standard error of the mean. P -values were calculated by two-way repeated measures analysis of variance (ANOVA) using Sidak’s post hoc test in GraphPad Prism 6 ( ∗∗∗ P

    Journal: Frontiers in Plant Science

    Article Title: Lack of S-RNase-Based Gametophytic Self-Incompatibility in Orchids Suggests That This System Evolved after the Monocot-Eudicot Split

    doi: 10.3389/fpls.2017.01106

    Figure Lengend Snippet: The lengths of the majority of D. chrysanthum pollen tubes present in compatible and incompatible styles at different times after pollination. Pollen tubes were measured at 12, 24, 48, 72, and 96 h after pollination. Each point is a mean value based on measurements of tubes in three styles, and the error bars represent ± the standard error of the mean. P -values were calculated by two-way repeated measures analysis of variance (ANOVA) using Sidak’s post hoc test in GraphPad Prism 6 ( ∗∗∗ P

    Article Snippet: Statistical analyses were performed using the software package GraphPad Prism 6 for Mac OS X (version 6.0c; GraphPad Software, Inc., La Jolla, CA, United States).

    Techniques:

    The lengths of the majority of D. longicornu pollen tubes present in compatible and incompatible styles at different times after pollination. Pollen tubes were measured at 2 D, 3 D, 4 D, 5 D, 7 D, and 9 D after pollination. Each point is a mean value based on measurements of tubes in three styles, and the error bars represent ± the standard error of the mean. P -values were calculated by two-way repeated measures analysis of variance (ANOVA) using Sidak’s post hoc -test in GraphPad Prism 6 ( ∗∗∗ P

    Journal: Frontiers in Plant Science

    Article Title: Lack of S-RNase-Based Gametophytic Self-Incompatibility in Orchids Suggests That This System Evolved after the Monocot-Eudicot Split

    doi: 10.3389/fpls.2017.01106

    Figure Lengend Snippet: The lengths of the majority of D. longicornu pollen tubes present in compatible and incompatible styles at different times after pollination. Pollen tubes were measured at 2 D, 3 D, 4 D, 5 D, 7 D, and 9 D after pollination. Each point is a mean value based on measurements of tubes in three styles, and the error bars represent ± the standard error of the mean. P -values were calculated by two-way repeated measures analysis of variance (ANOVA) using Sidak’s post hoc -test in GraphPad Prism 6 ( ∗∗∗ P

    Article Snippet: Statistical analyses were performed using the software package GraphPad Prism 6 for Mac OS X (version 6.0c; GraphPad Software, Inc., La Jolla, CA, United States).

    Techniques: